We wanted to check the validity of the gun’s design by testing if it could generate pressure pulses which would penetrate a plant cell wall. This was done using onion epidermal tissue, as the tissue layers are only one cell thick so would be the easiest to observe any penetration on microscope imaging.

For bombardment, we prepared the macrocarriers as described in the gun assembly protocol, steps 14-15. We then used simple agar TAP plates we had spare in the lab (approximately 1cm thick), and cut 2x1cm strips of brown onion epidermis to place on the plates. (Figure 1: We initially coloured the plates purple as we thought this would make imaging the onion epidermis easier to observe, but it was not necessary)

We then pipetted 150mg of M10 tungsten microparticles, prepared according to our wetlab biolistics protocol, onto each macrocarrier. A fresh 16g CO2 cartridge lasted for around 20 bombardments, ranging from 130-155 psi (though the efficiency of this improved significantly once we became more experienced at pressurising the gun and wasted less CO2).

On inspecting the bombarded onion samples under the microscope, we found evidence of holes in the onion tissue. This suggests these pressures could be suitable for biolistics on plant cells, as the tungsten microparticles were capable of penetrating plant cell walls so should penetrate cells for biolistic transformation.

Our next step in developing the gene gun would be experiment with lower pressures, to find the most efficient pressure pulse for penetration of cells. In the future, we also aim to prove the gun’s use in carrying out chloroplast transformations, by attempting transformation of chlamydomonas samples.

Gene Gun

We have managed to create a low-cost, DIY, gene gun that would fire DNA into plant cells. The complete assembly instructions, materials required, and an example of it working under real world condition can be found here

Cas9 Modelling

We managed to write a code that would enable anyone to predict the time taken to achieve homoplasmy using the appropriate model for Cas9 activity. This program is also designed to be flexible in the sense that various input parameters can be changed to fit the parameters of your experiment. For more information, please read a complete documentation of the program under the 'Modeling' page.